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Policymakers aim to move toward animal-free alternatives for scientific research and have introduced very strict regulations for animal research. We argue that, for neuroscience research, until viable and translational alternatives become available and the value of these alternatives has been proven, the use of animals should not be compromised., We would like to thank Loren Frank, UCSF, USA; Sheena Josselyn, Hospital for Sick Children, University of Toronto, Canada; Shantanu Jadhav, Brandeis University, USA; the European Animal Research Association (EARA); the Federation of European Neuroscience Societies Committee on Animals in Research (CARE); the Swiss Society for Neuroscience; the Society for Neuroscience Committee on Animals in Research (CAR); and Stichting Informatie Dierproeven (the Dutch foundation for public information on animal testing: SID) for input on and support for this article.

Rapastinel is a novel psychoactive substance that acts as an N-methyl-D-aspartate-receptor (NMDAR) agonist and triggers antidepressant- and antipsychotic-like effects in animal models. However, it is unknown if rapastinel possesses a better side-effect profile than fast-acting glutamatergic antidepressants, like ketamine, which trigger neurotoxicity in the perinatal rodent cortex and protracted schizophrenia-like alterations. Here we found a remarkable neuroprotective effect of rapastinel against apoptosis induced by the NMDAR antagonist MK-801 in comparison to that elicited by clozapine and the mGlu2/3 agonist LY354740. These results suggest the potential therapeutic/prophylactic effect of rapastinel in ameliorating deleterious effects induced by NMDAR blockade during neurodevelopment.

This paper aims to review data regarding two determinants of comorbidity between depression and obesity, i.e., the role of disturbed gut microbiome in their genesis and of diets in their treatment. Obesity and major depressive disorders (MDD) are highly comorbid, the “metabolic” (obese) subtype of MDD affects about one third of all individuals with MDD. There is an urgent need for better therapies strategies, which may include specific dietary measures. A diet low in carbohydrates (low-carb), effective in obesity, may be beneficial also in MDD. However, the underlying mechanisms have not yet been elucidated. Recent data suggest a key role of gut microbiota, neuroplasticity, and neuroinflammation in obesity and MDD. We will focus on the brain-derived neurotrophic factor (BDNF), and microglial fractalkine, a main modulator of neuroinflammation. BDNF and fractalkine may be involved in “metabolic” depression. Future studies may uncover specific pathophysiological pathways in affected patients towards more efficient causal therapies.

Silencing of dopamine transporter (DAT), a main controlling factor of dopaminergic signaling, results in biochemical and behavioral features characteristic for neuropsychiatric diseases with presumed hyperdopaminergia including schizophrenia, attention deficit hyperactivity disorder (ADHD), bipolar disorder, and obsessive-compulsive disorder (OCD). Investigation of DAT silencing thus provides a transdiagnostic approach towards a systems-level understanding of common underlying pathways. Using a high-field multimodal imaging approach and a highly sensitive cryogenic coil, we integrated structural, functional and metabolic investigations in tandem with behavioral assessments on a newly developed preclinical rat model, comparing DAT homozygous knockout (DAT-KO, N = 14), heterozygous knockout (N = 8) and wild-type male rats (N = 14). We identified spatially distributed structural and functional brain alterations encompassing motor, limbic and associative loops that demonstrated strong behavioral relevance and were highly consistent across imaging modalities. DAT-KO rats manifested pronounced volume loss in the dorsal striatum, negatively correlating with cerebellar volume increase. These alterations were associated with hyperlocomotion, repetitive behavior and loss of efficient functional small-world organization. Further, prefrontal and midbrain regions manifested opposite changes in functional connectivity and local network topology. These prefrontal disturbances were corroborated by elevated myo-inositol levels and increased volume. To conclude, our imaging genetics approach provides multimodal evidence for prefrontal-midbrain decoupling and striato-cerebellar neuroplastic compensation as two key features of constitutive DAT blockade, proposing them as transdiagnostic mechanisms of hyperdopaminergia. Thus, our study connects developmental DAT blockade to systems-level brain changes, underlying impaired action inhibition control and resulting in motor hyperactivity and compulsive-like features relevant for ADHD, schizophrenia and OCD.

Extensive evidence suggests a dysfunction of the glutamate NMDA receptor (NMDAR) in schizophrenia, a severe psychiatric disorder with putative early neurodevelopmental origins, but clinical onset mainly during late adolescence. On the other hand, pharmacological models using NMDAR antagonists and the clinical manifestation of anti-NMDAR encephalitis indicate that NMDAR blockade/hypofunction can trigger psychosis also at adult stages, without any early developmental dysfunction. Previous genetic models of NMDAR hypofunction restricted to parvalbumin-positive interneurons indicate the necessity of an early postnatal impairment to trigger schizophrenia-like abnormalities, whereas the cellular substrates of NMDAR-mediated psychosis at adolescent/adult stages are unknown. Neuregulin 1 (NRG1) and its receptor ErbB4 represent schizophrenia-associated susceptibility factors that closely interact with NMDAR. To determine the neuronal populations implicated in “late” NMDAR-driven psychosis, we analyzed the effect of the inducible ablation of NMDARs in ErbB4-expressing cells in mice during late adolescence using a pharmacogenetic approach. Interestingly, the tamoxifen-inducible NMDAR deletion during this late developmental stage did not induce behavioral alterations resembling depression, schizophrenia or anxiety. Our data indicate that post-adolescent NMDAR deletion, even in a wider cell population than parvalbumin-positive interneurons, is also not sufficient to generate behavioral abnormalities resembling psychiatric disorders. Other neuronal substrates that have to be revealed by future studies, may underlie post-adolescent NMDAR-driven psychosis.

Soy is one of the most common sources of protein in many commercial formulas for laboratory rodent diets. Soy contains isoflavones, which are estrogenic. Therefore, soy-containing animal diets might influence estrogen-regulated systems, including basal behavioral domains, as well as affective behavior and cognition. Furthermore, the isoflavone content of soy varies, potentially unpredictably confounding behavioral results. Therefore researchers are increasingly considering completely avoiding dietary soy to circumvent this problem. Several animal studies have investigated the effects of soy free diets but produced inconsistent results. In addition, most of these previous studies were performed in outbred rat or mouse strains. In the current study, we assessed whether a soy-free diet altered locomotion, exploration, nesting, anxiety-related behaviors, learning, and memory in C57BL/6 mice, the most common inbred strain used in biomedical research. The parameters evaluated address measures of basic health, natural behavior, and affective state that also are landmarks for animal welfare. We found minor differences between feeding groups but no indications of altered welfare. We therefore suggest that a soy-free diet can be used as a standard diet to prevent undesirable side effects of isoflavones and to further optimize diet standardization, quality assurance, and ultimately increase the reproducibility of experiments.

Animal models in psychiatric research are indispensable for insights into mechanisms of behaviour and mental disorders. Distress is an important aetiological factor in psychiatric diseases, especially depression, and is often used to mimic the human condition. Modern bioethics requires balancing scientific progress with animal welfare concerns. Therefore, scientifically based severity assessment of procedures is a prerequisite for choosing the least compromising paradigm according to the 3Rs principle. Evidence-based severity assessment in psychiatric animal models is scarce, particularly in depression research. Here, we assessed severity in a cognitive depression model by analysing indicators of stress and well-being, including physiological (body weight and corticosterone metabolite concentrations) and behavioural (nesting and burrowing behaviour) parameters. Additionally, a novel approach for objective individualised severity grading was employed using clustering of voluntary wheel running (VWR) behaviour. Exposure to the paradigm evoked a transient elevation of corticosterone, but neither affected body weight, nesting or burrowing behaviour. However, the performance in VWR was impaired after recurrent stress exposure, and the individual severity level increased, indicating that this method is more sensitive in detecting compromised welfare. Interestingly, the direct comparison to a somatic, chemically induced colitis model indicates less distress in the depression model. Further objective severity assessment studies are needed to classify the severity of psychiatric animal models in order to balance validity and welfare, reduce the stress load and thus promote refinement.

[This corrects the article DOI: 10.1371/journal.pone.0240439.].

The translational assessment of mechanisms underlying cognitive functions using touchscreen-based approaches for rodents is growing in popularity. In these paradigms, daily training is usually accompanied by extended food restriction to maintain animals' motivation to respond for rewards. Here, we show a transient elevation in stress hormone levels due to food restriction and touchscreen training, with subsequent adaptation effects, in fecal corticosterone metabolite concentrations, indicating effective coping in response to physical and psychological stressors. Corticosterone concentrations of experienced but training-deprived mice revealed a potential anticipation of task exposure, indicating a possible temporary environmental enrichment-like effect caused by cognitive challenge. Furthermore, the analyses of immediate early gene (IEG) immunoreactivity in the hippocampus revealed alterations in Arc, c-Fos and zif268 expression immediately following training. In addition, BDNF expression was altered as a function of satiation state during food restriction. These findings suggest that standard protocols for touchscreen-based training induce changes in hippocampal neuronal activity related to satiation and learning that should be considered when using this paradigm.

Minocycline is a broad-spectrum tetracycline antibiotic with multiple actions, including anti-inflammatory and neuroprotective effects, that was proposed as novel treatment for several psychiatric disorders including schizophrenia and depression. However, there are contradictory results regarding antidepressant effects of minocycline in rodent models. Additionally, the possible anxiolytic effect of minocycline is still poorly investigated. Therefore, we aimed to clarify in the present study the influence of minocycline on behavioral correlates of mood disorders in standard tests for depression and anxiety, the Porsolt Forced Swim Test (FST), Elevated O-Maze, Dark-Light Box Test and Openfield Test in adult C57BL/6 mice. We found, unexpectedly, that mice treated with minocycline (20-40mg/kg, i.p.) did not display antidepressant- or anxiolytic-like behavioral changes in contrast to mice treated with diazepam (0.5mg/kg, anxiety tests) or imipramine (20mg/kg, depressive-like behavior). These results are relevant for future studies, considering that C57BL/6 mice, the most widely used strain in pharmacological and genetic animal models, did not react as expected to the treatment regime applied.

Early life stress compromises brain development and can contribute to the development of mental illnesses. A common animal model used to study different facets of psychiatric disorders is social isolation from early life on. In rats, this isolation can induce long-lasting alterations in molecular expression and in behavior. Since social isolation models severe psychiatric symptoms, it is to be expected that it affects the overall wellbeing of the animals. As also promoted by the 3Rs principle, though, it is pivotal to decrease the burden of laboratory animals by limiting the number of subjects (reduce, replace) and by improving the animals' wellbeing (refine). The aim of this study was therefore to test possible refinement strategies such as resocialization and mere adult social isolation. We examined whether the alternatives still triggered the necessary phenotype while minimizing the stress load on the animals. Interestingly, we did not find reduced wellbeing-associated burrowing performance in isolated rats. The hyperactive phenotype seen in socially isolated animals was observed for rats undergoing the adult-only isolation, but resocializing ameliorated the locomotor abnormality. Isolation strongly affected markers of neuroplasticity in the prefrontal cortex independent of timing: mRNA levels of Arc, Bdnf and the pool of Bdnf transcripts with the 3' long UTR were reduced in all groups. Bdnf splice variant IV expression was reduced in lifelong-isolated animals. Some of these deficits normalized after resocialization; likewise, exon VI Bdnf mRNA levels were reduced only in animals persistently isolated. Conversely, social deprivation did not affect the expression of Gad67 and Pvb, two GABAergic markers, whereas changes occurred in the expression of dopamine d1 and d2 receptors. As adult isolation was sufficient to trigger the hyperactive phenotype and impaired neuroplasticity in the prefrontal cortex, it could be a candidate for a refinement strategy for certain research questions. To fully grade the severity of post-weaning social isolation and the alternatives, adult isolation and resocialization, a more profound and multimodal assessment approach is necessary.

Background The glutamate hypothesis of schizophrenia postulates NMDA receptor (NMDAR) hypofunction as a major pathophysiological mechanism based on clinical observations and preclinical evidence. Mice with globally reduced NMDAR expression display hyperlocomotion, sensorimotor gating deficits, cognitive deficits and social withdrawal. NMDARs are tetramers composed of 2 obligatory GluN1 (NR1) and 2 variable GluN2A-D (NR2A-D) subunits. GluN2A and GluN2B are the main cortical NMDAR subunits, but genetic models provided evidence that psychosis-like effects of NMDAR antagonists are mediated by GluN2C/GluN2D subunits. Previous data suggest a role also of the GluN2D subunit in schizophrenia-associated abnormalities [1]. Methods We aimed to assess a comprehensive behavioural phenotyping of GluN2D deficient mice, including locomotion, affective behaviours and general welfare, but drew special focus on cognitive functions, including spatial learning, short-term and long-term memory, as well as puzzle solving. We further investigated the effect of D-Serine on the GluN2D KO mice. D-Serine is a NMDAR modulator and acts as an agonist at the glycine site. Previous work in clinical trials suggests significant therapeutic effects of D-serine on negative and positive symptoms, cognitive deficits and motor symptoms. We used the chronic application of D-Serin by supplementing it into the drinking water of the subjects. Results Preliminary data indicate no effect on locomotor alterations of the GluN2D mice, however a potential improvement in short-term memory and wellbeing-associated parameters. They indicate a role of the GluN2D subunit and a beneficial effect of D-serine on schizophrenia-induced parameters, including physiological, welfare-associated burrowing and cognitive performance in the short term task of the puzzle box test. Discussion Our preliminary data indicate a role of the GluN2D subunit in the emergence of cognitive deficits associated with schizophrenia and a potential therapeutic effect of D-serine in alleviating these deficits. Further investigations are planned to further validate these findings. Reference

A growing body of evidence supports the idea that drugs targeting the glutamate system may represent a valuable therapeutic alternative in major depressive disorders (MDD). The rapid and prolonged mood elevating effect of the NMDA receptor (NMDAR) antagonist ketamine has been studied intensely. However, its clinical use is hampered by deleterious side-effects, such as psychosis. Therefore, a better understanding of the mechanisms of the psychotropic effects after NMDAR blockade is necessary to develop glutamatergic antidepressants with improved therapeutic profile. Here we review recent experimental data that addressed molecular/cellular determinants of the antidepressant effect mediated by inactivating NMDAR subtypes. We refer to results obtained both in pharmacological and genetic animal models, ranging from global to conditional NMDAR manipulation. Our main focus is on the contribution of different NMDAR subtypes to the psychoactive effects induced by NMDAR ablation/blockade. We review data analyzing the effect of NMDAR subtype deletions limited to specific neuronal populations/brain areas in the regulation of mood. Altogether, these studies suggest effective and putative specific NMDAR drug targets for MDD treatment.

The brain-derived neurotrophic factor (BDNF) is a major proliferative agent in the nervous system. Both BDNF-deficiency and perinatal hypoxia represent genetic/environmental risk factors for schizophrenia. Moreover, a decreased BDNF response to birth hypoxia was associated with the disease. BDNF expression is influenced by neuronal activity and environmental conditions such as hypoxia. Thus, it may partake in neuroprotective and reparative mechanisms in acute or chronic neuronal insults. However, the interaction of hypoxia and BDNF is insufficiently understood and the behavioral outcome unknown. Therefore, we conducted a battery of behavioral tests in a classical model of chronic early postnatal mild hypoxia (10% O2), known to significantly impair brain development, in BDNF-deficient mice. We found selective deficits in measures associated with sensorimotor gating, namely enhanced acoustic startle response (ASR) and reduced prepulse inhibition (PPI) of ASR in BDNF-deficient mice. Unexpectedly, the alterations of sensorimotor gating were caused only by BDNF-deficiency alone, whereas hypoxia failed to evoke severe deficits and even leads to a milder phenotype in BDNF-deficient mice. As deficits in sensorimotor gating are present in schizophrenia and animal models of the disease, our results are of relevance regarding the involvement of BDNF in its pathogenesis. On the other hand, they suggest that the effect of perinatal hypoxia on long-term brain abnormalities is complex, ranging from protective to deleterious actions, and may critically depend on the degree of hypoxia. Therefore, future studies may refine existing hypoxia protocols to better understand neurodevelopmental consequences associated with schizophrenia.

Background To explore the domain-general risk factor of early-life social stress in mental illness, rearing rodents in persistent postweaning social isolation has been established as a widely used animal model with translational relevance for neurodevelopmental psychiatric disorders such as schizophrenia. Although changes in resting-state brain connectivity are a transdiagnostic key finding in neurodevelopmental diseases, a characterization of imaging correlates elicited by early-life social stress is lacking. Methods We performed resting-state functional magnetic resonance imaging of postweaning social isolation rats (N = 23) 9 weeks after isolation. Addressing well-established transdiagnostic connectivity changes of psychiatric disorders, we focused on altered frontal and posterior connectivity using a seed-based approach. Then, we examined changes in regional network architecture and global topology using graph theoretical analysis. Results Seed-based analyses demonstrated reduced functional connectivity in frontal brain regions and increased functional connectivity in posterior brain regions of postweaning social isolation rats. Graph analyses revealed a shift of the regional architecture, characterized by loss of dominance of frontal regions and emergence of nonfrontal regions, correlating to our behavioral results, and a reduced modularity in isolation-reared rats. Conclusions Our result of functional connectivity alterations in the frontal brain supports previous investigations postulating social neural circuits, including prefrontal brain regions, as key pathways for risk for mental disorders arising through social stressors. We extend this knowledge by demonstrating more widespread changes of brain network organization elicited by early-life social stress, namely a shift of hubness and dysmodularity. Our results highly resemble core alterations in neurodevelopmental psychiatric disorders such as schizophrenia, autism, and attention-deficit/hyperactivity disorder in humans.

Behavioral experiments are usually designed to tap into a specific cognitive function, but animals may solve a given task through a variety of different and individual behavioral strategies, some of them not foreseen by the experimenter. Animal learning may therefore be seen more as the process of selecting among, and adapting, potential behavioral policies, rather than mere strengthening of associative links. Calcium influx through high-voltage-gated Ca2+ channels is central to synaptic plasticity, and altered expression of Cav1.2 channels and the CACNA1C gene have been associated with severe learning deficits and psychiatric disorders. Given this, we were interested in how specifically a selective functional ablation of the Cacna1c gene would modulate the learning process. Using a detailed, individual-level analysis of learning on an operant cue discrimination task in terms of behavioral strategies, combined with Bayesian selection among computational models estimated from the empirical data, we show that a Cacna1c knockout does not impair learning in general but has a much more specific effect: the majority of Cacna1c knockout mice still managed to increase reward feedback across trials but did so by adapting an outcome-based strategy, while the majority of matched controls adopted the experimentally intended cue-association rule. Our results thus point to a quite specific role of a single gene in learning and highlight that much more mechanistic insight could be gained by examining response patterns in terms of a larger repertoire of potential behavioral strategies. The results may also have clinical implications for treating psychiatric disorders., Author summary To deal with an uncertain and complex world, animals have developed a large repertoire of behavioral heuristics and default strategies that spring into action in unknown situations. Building on this a priori repertoire, animals may find various ways to succeed on a given behavioral task. Therefore, determining the exact behavioral strategy followed during a task may be essential for understanding the cognitive processes involved. Using computational models to analyze behavior, we examined how a genetic variation in a gene that encodes a calcium channel and has been associated with learning deficits influences the way in which animals acted on a task in which a reward was associated with a specific behavior. We found that a knockout of the relevant gene does not lead to a general learning impairment but rather led animals to adopt a behavioral strategy different from the one employed by the control animals. Specifically, knockout animals managed to increase their reward returns by basing their responses more on the previous reward location rather than on reward-indicating stimuli, like the controls did. These findings may prove useful for behavioral therapy in the context of psychiatric disorders associated with this specific gene variation.

Depression is a highly heterogeneous disorder presumably caused by a combination of several factors ultimately causing the pathological condition. The genetic liability model of depression is likely to be of polygenic heterogeneity. miRNAs can regulate multiple genes simultaneously and therefore are candidates that align with this model. The habenula has been linked to depression in both clinical and animal studies, shifting interest towards this region as a neural substrate in depression. The goal of the present study was to search for alterations in miRNA expression levels in the medial and lateral habenula of rats exposed to the learned helplessness (LH) rat model of depression. Ten miRNAs showed significant alterations associating with their response to the LH paradigm. Of these, six and four miR-NAs were significantly regulated in the MHb and LHb, respectively. In the MHb we identified miR-490, miR-291a-3p, MiR-467a, miR-216a, miR-18b, and miR-302a. In the LHb miR-543, miR-367, miR-467c, and miR-760-5p were significantly regulated. A target gene analysis showed that several of the target genes are involved in MAPK signaling, neutrophin signaling, and ErbB signaling, indicating that neurotransmission is affected in the habenula as a consequence of exposure to the LH paradigm.